Vanillin, whose chemical name is 4-hydroxy-3-methoxybenzaldehyde, is one of the most important aromatic flavor compound used in food, beverages, fragrances and phaimaceuticals. Vanillin was historically extracted from Vanilla planifolia, Vanilla tahitiensis and Vanilla pompona pods. Today, as a result of constantly rising demand, less than 5% of worldwide vanillin production comes from vanilla orchid. Currently, chemical synthesis is the most important process for producing vanillin. However, there is a growing interest in other sources of vanillin and in particular in bio-based routes using bioconversion processes from natural raw material. The use of microbial cells and their enzymes as biocatalysts for the synthesis of chemicals and flavor compounds has attracted much attention lately. Advantageously, the products of such bioconversions are considered as ‘natural products’ by the European Community Legislation.
Bioconversion processes are based on the following substrates: lignin, phenolic stilbenes, isoeugenol, eugenol, ferulic acid, sugars, aromatic amino acids and waste residues containing these precursors. A recent review (Kaur B, Chakraborty D. “Biotechnological and molecular approaches for vanillin production: a review” Appl Biochem Biotechnol. 2013 February; 169(4):1353-72) lists several biosynthetic pathways and appropriate microorganisms used for biosynthesis of vanilloids.
Strains of the genus Amycolatopsis have been identified as being able to synthetize vanillin from ferulic acid, a natural cell wall component of higher plants (U.S. Pat. No. 6,133,003). Among the strains from this genus, the strain accessible under number ATCC 39116 has been selected as being capable of synthetizing large amounts of vanillin, due to its high resistance to vanillin toxicity.
The metabolic pathway of conversion of ferulic acid into vanillin is shown in FIG. 1. In wild-type Amycolatopsis strains, the produced vanillin is then converted into both vanillic acid and vanillic alcohol, as shown in FIG. 2. This unwanted consumption of vanillin by endogenous enzymes is a major obstacle for using microorganisms of the Actinomycetales order in an industrialized process for producing vanillin.
In order to prevent the oxidation of vanillin into vanillic acid, the gene coding for the enzyme responsible of this oxidation reaction entitled vanillin dehydrogenase has been recently investigated in Amycolatopsis sp. strain ATCC39116. A putative vdh gene was identified, characterized and a vdh deletion mutant was generated. Fermentation of this mutant strain enables the obtaining of a 2.3-fold higher vanillin concentration, compared to fermentation of the wild-type strain, and a substantially reduced amount of vanillic acid was observed (Fleige C, Hansen G, Kroll J and Steinbüchel A, Investigation of the Amycolatopsis sp. strain ATCC 39116 vanillin dehydrogenase and its impact on the biotechnical production of vanillin, Appl. Environ. Microbial. 2013, vol. 79, 81; patent application WO 2012/172108). The vdh gene is accessible in NCBI database, under accession number AFY98904.
The conversion of vanillin into vanillic alcohol is catalysed by an enzyme having vanillin reductase activity. However, this enzyme has not been identified yet in a strain of Amycolatopsis sp. neither in any strain of the order of Actinomycetales.